In the food and medical fields, methods for efficiently purifying useful proteins are strongly desired. In the biotechnological industry, large-scale purification of proteins has represented an important challenge in recent years. Particularly in the pharmaceutical field, the demand for antibody drugs is rapidly growing; there is a strong desire to establish technologies that make it possible to efficiently produce and purify large amounts of proteins.
Generally, proteins are produced by cell cultures using animal-derived cell lines. In a typical operation to purify a protein from a cell culture, the cell culture is first centrifuged to precipitate and remove turbid components. Then, cell debris having a size of about 1 μm or less that can not be completely removed by the centrifugation are removed by means of size filtering using a microfiltration membrane. Further, the filtrate is subjected to sterilization filtration using a filtration membrane having a maximum pore size of 0.22 μm or less for sterilization to provide a sterilized solution containing a desired protein (a harvest step). Subsequently, using a purification process employing a combination of a plurality of chromatographic techniques including affinity chromatography typically using protein A, the desired protein is separated and purified by removing contaminants such as host cell proteins (HCPs), deoxyribonucleic acids (DNAs), aggregates of the desired protein, endotoxins, viruses, protein A detached from the column, and aggregates of protein A and antibodies from the sterilized solution (a downstream step).
The concentration of a desired protein in a cell culture subjected to a conventional purification method for a protein as described above is now typically on the order of 1 g/L. The concentration of contaminants is also probably almost comparable to or less than the concentration of the desired protein. In such a concentration, even a conventional purification method for a protein comprising the harvest step and the downstream step can be useful.
However, because the demand for antibody drugs rapidly grows and the production of proteins used in the antibody drugs has been large-scale-oriented, cell culture technologies for increasing the concentration of proteins in cell cultures have been rapidly advanced in recent years. Thus, the concentration of desired proteins in cell cultures sometimes reaches 10 g/L or more. At the same time, the concentration of contaminants in the cell cultures similarly increases; it is becoming difficult to purify desired proteins by conventional purification methods for proteins.
Particularly, an increased concentration of a desired antibody protein in a cell culture also tends to noticeably increase the concentration of aggregates of its monomer, for example, multimers such as a dimer and a trimer. Such aggregates can cause complement activation or anaphylaxis when they are administered into a living body. It is therefore pointed out that the aggregates can deleteriously affect the safety of the antibody drug; an effective method for the removal thereof has been strongly desired in recent years. For solving the problem, chromatography processes are reported in large numbers which aim to effectively remove various contaminants including the aggregates to purify antibody proteins used as antibody drugs, namely a monoclonal antibody, a polyclonal antibody, a humanized antibody, a human antibody, an immunoglobulin, and the like.
Ion-exchange chromatography is a method for separating an antibody and contaminants by utilizing the difference of their isoelectric points. Particularly, anion-exchange chromatography is frequently used for removing contaminants such as HCP, DNA and virus generally having lower isoelectric points than antibody proteins. A method for purifying an antibody monomer by removing aggregates having isoelectric points almost equal to that of the monomer is also proposed.
For a method for removing common contaminants such as HCP, protein adsorption membranes to which protein adsorption capability is imparted by introducing ion-exchange groups into the porous membrane have recently been developed (see, for example, Patent Literatures 1 and 2). As a usage example of protein adsorption membranes, Patent Literature 3 also discloses a method for separating albumin from a lymph fluid using two types of protein adsorption membranes, i.e. a porous cellulose membrane into which anion-exchange groups are introduced and a porous cellulose membrane into which cation-exchange groups are introduced. Further, Patent Literature 4 discloses a method for separating nucleic acid and endotoxin using a porous cellulose membrane into which anion-exchange groups are introduced. Further, Patent Literatures 5 and 6 also disclose protein adsorption membranes in each of which cation-exchange groups and anion-exchange groups are introduced into a porous polyether sulfone membrane.
In addition, Patent Literature 7 discloses a porous membrane having a swollen gel layer in which a primary amine is immobilized as an anion-exchange group, as a porous membrane suitable for purifying an antibody monomer by removing impurities from a solution in a state of a relatively high salt concentration, that is, of high electric conductivity, such as a cell culture or an eluate of a cation-exchange chromatography step.
As a method for removing antibody aggregates, for example, Patent Literature 8 also discloses a purification method for an antibody monomer, which involves adjusting a mixed solution of the antibody monomer and aggregates to a pH near the isoelectric point of the antibody, passing the mixed solution through an anion-exchange chromatography column and recovering the passed solution, further passing buffer solutions of the same pH therethrough and recovering the wash solutions, and using these recovered solutions as purified solutions of the antibody monomer. This purification method is based on the principle that the aggregate is easily, although slightly, immobilized by anion-exchange groups compared to the monomer, because it has more charge points than the monomer. Patent Literature 9 also discloses a purification method for an antibody monomer, which involves adsorbing the antibody monomer and aggregates to an anion-exchange chromatography column and performing gradient elution in which the salt concentration in the eluate is gradually increased to collect the elution peak fraction of the antibody monomer first eluted.
It has been widely studied in recent years to use a chromatography column packed with mixed-mode resins having a plurality of ligands in order to efficiently separate and purify an antibody from contaminants. Ligands called mixed mode ligands or multi-modal ligands are used in such a chromatography. Chromatography using such ligands is expected to be capable of simultaneously and effectively removing a plurality of contaminants as well as enabling higher-precision separation by utilizing a plurality of differences in interactions: a difference in charge interaction and a difference in hydrophobic interaction.
For example, Patent Literature 10 describes a method which involves adsorbing contaminants and recovering an antibody monomer in a flow-through mode using chromatography with multi-modal ligands consisting of an anion-exchange group and a hydrophobic group, particularly a method which involves adsorbing and removing an aggregate consisting of a liberated protein A ligand and an antibody monomer. Patent Literature 11 also describes a method which involves adsorbing most contaminants such as DNA, virus, endotoxin, aggregates, and HCP using chromatography with multi-modal ligands having a quaternary ammonium group, a hydrogen-bonding group, and a hydrophobic group and recovering an antibody monomer by flow-through, and particularly describes that HCP is almost completely removed. In addition, Patent Literature 12 describes purifying an antibody monomer in a binding mode by using chromatography with mixed mode ligands having a mercapto group and an aromatic pyridine ring to adsorb only the antibody monomer and remove aggregates as non-adsorption fractions.
Patent Literature 13 describes an example in which chromatography with hydroxyapatite was applied as a method for purifying and recovering an antibody monomer in a flow-through mode intended for selectively adsorbing aggregates to a column and more effectively removing the aggregates. Patent Literature 7 also describes a porous membrane in which an anion-exchange group using an amino group is immobilized on the surface of a substrate via graft chains, and discloses a method for immobilizing limited amino groups by a gas phase reaction. In addition, Patent Literature 8 describes a method for separating an egg-white protein using a porous membrane on which diethylamino groups and 2-hydroxyethylamino groups are immobilized via graft chains.